P1235: INVESTIGATION OF THE CO-MUTATIONAL LANDSCAPE OF ALK-POSITIVE ALCL
Authors: Matteo Villa, Federica Malighetti, Mario Mauri, Geeta G Sharma, Cosimo Lobello, Hugo Larose, Alessandra Pirola, Silvia Bombelli, Nicoletta Cordani, Luca Massimino, Šárka Pospíšilová, Suzanne D. Turner, Giorgio Inghirami, Lisa Pagani, Lucrezia Criscuolo, Fulvio Magni, Roberto Perego, Fabio Pagni, Rocco Piazza, Roberto Chiarle, Carlo Gambacorti-Passerini, Luca Mologni
Journal: HemaSphere (2023)

Topic: 20. Lymphoma Biology & Translational Research Background: Anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) is an aggressive lymphoma characterized by expression of the NPM::ALK fusion tyrosine kinase essential for neoplastic cells growth. If the disease becomes resistant or relapses after initial chemotherapy, the prognosis for ALK+ ALCL is bleak. Despite being primarily driven by the constantly active NPM::ALK kinase, the presence of other mutations can influence the course of the disease and its response to treatment, resulting in varying outcomes among patients. Aims: The aim of this project is the identification and characterization of additional genetic abnormalities that may co-exist along with the ALK fusion gene in ALCL patients, through Next-Generation Sequencing (NGS) technology. Methods: Diagnosis biopsy and matched healthy control DNA samples from 24 ALK+ ALCL patients were sequenced. Analysis of whole-exome sequencing (WES) raw data was performed using our pipeline and somatic variants were annotated (≥25% variant allele frequency in the tumor and ≤5% in the matched healthy tissue) that could be potential co-drivers. All variants were validated through Sanger sequencing. The identified mutations were characterized in cell models to analyse their oncogenic potential and impact on protein function. Results: The analysis confirmed our hypothesis that ALK+ ALCL carry additional somatic variants. Gene ontology and pathway analyses of the identified mutated genes pointed to cell movement-related and cancer-related terms. The most attracting genes were FAT4 and RUNX1T1 since they exhibited a higher frequency (13% and 8%, respectively) among ALK+ ALCL patients and held potential functional significance. FAT4 is a transmembrane receptor belonging to the cadherin family with tumor suppressor functions. FAT4 mutations identified in our patients cohort were predicted to have a loss of function effect. We created FAT4 knockdown ALCL cell lines (siFAT4) that showed increased growth rates and greater migratory abilities in comparison to the parental cells. They displayed different morphology, characterized by larger size and altered rearrangement of F-actin. siFAT4 cells had reduced levels of Ser/Thr phosphorylated β-catenin (S33/S37/T41) and increased nuclear localization, suggesting the activation of the Wnt/β-catenin signaling pathway and consequent promotion of cellular proliferation. Indeed, siFAT4 cells show upregulation of the target genes of this pathway. We observed a decreased phosphorylation of YAP1 (S127) protein in siFAT4 cells in comparison to the parental cells, indicating inactivation of the Hippo pathway. Moreover, we observed in siFAT4 cells an increased phosphorylation of proteins involved in proliferation, such as STAT3 and STAT1. Finally, siFAT4 cells were less sensitive to CHOP treatment compared to parental cells. RUNX1T1 acts as a transcriptional corepressor. Mutations increased the repressor activity of RUNX1T1 in reporter assays. We generated RUNX1T1 mutated ALCL cell lines, which appeared to have greater migration than the WT form. In both projects, RNA-Seq was carried out to compare the silenced/mutated cell lines with their parental counterparts. Consistent with previous findings from patient samples, terms related to cancer and cell movement were prominently enriched. Summary/Conclusion: Collectively, the data we have gathered sheds light on genes, apart from NPM::ALK, that may have a role in the development of ALCL. Keywords: Drug resistance, ALK/ALCL, Gene mutation